Several scoring systems have been used to predict short-term outcome in patients with out-of-hospital cardiac arrest (OHCA), including the disease-specific OHCA and CAHP  scores, as well as the general severity-of-illness scores Acute Physiology and Chronic Health Evaluation II (APACHE II) and Simplified Acute Physiology Score II (SAPS II). This study aimed to assess the prognostic performance of these four scores to predict long-term outcomes (≥ 2 years) in adult cardiac arrest patients.This is a prospective single-centre cohort study including consecutive cardiac arrest patients admitted to intensive care in a Swiss tertiary academic medical centre. The primary endpoint was 2-year mortality. Secondary endpoints were neurological outcome at 2 years post-arrest assessed by Cerebral Performance Category with CPC 1–2 defined as good and CPC 3–5 as poor neurological outcome, and 6-year mortality.In 415 patients admitted to intensive care, the 2-year mortality was 58.1%, with 96.7% of survivors showing good neurological outcome. The 6-year mortality was 82.5%. All four scores showed good discriminatory performance for 2-year mortality, with areas under the receiver operating characteristics curve (AUROC) of 0.82, 0.87, 0.83 and 0.81 for the OHCA, CAHP, APACHE II and SAPS II scores. The results were similar for poor neurological outcome at 2 years and 6-year mortality.This study suggests that two established cardiac arrest-specific scores and two severity-of-illness scores provide good prognostic value to predict long-term outcome after cardiac arrest and thus may help in early goals-of-care discussions.The online version contains supplementary material available at 10.1186/s13613-022-01048-y. Cardiac arrest is a global public health challenge and an important cause of premature death , 2. MortIn the past 15 years, several clinical risk scores have been developed and validated specifically for early prognostication after cardiac arrest , 23. StiThis study aims to evaluate the performance of the OHCA, CAHP, APACHE II, and SAPS II scores for early prognostication of long-term mortality and long-term neurological outcome in a large-scale prospective cohort of cardiac arrest patients.www.eknz.ch) and followed the principles of the Declaration of Helsinki and its amendments. Analysis and reporting for this study were conducted in accordance with the Transparent Reporting of a Multivariable Prediction Model for Individual Prognosis or Diagnosis (TRIPOD) statement [This study was conducted using the prospective COMMUNICATE/PROPHETIC cohort of consecutive cardiac arrest patients admitted to the 42-bed interdisciplinary ICU of the University Hospital Basel, Switzerland . The COMMUNICATE/PROPHETIC study investigates the outcomes of cardiac arrest patients and the psychosocial stress of their relatives. The details of the study conductance have been published previously , 36–41. Between October 2012 and November 2019, all patients with ROSC admitted to the ICU after OHCA or in-hospital cardiac arrest (IHCA) were prospectively included. Not eligible were patients suffering a cardiac arrest while being monitored . Further exclusion criteria were age  < 16 years or denial of informed consent. The patients were treated according to the standardised local treatment protocol, including targeted temperature management, in compliance with the guidelines of the European Resuscitation Council , 43, 44.The primary outcome was long-term mortality at 2 years. Secondary outcomes were long-term neurological outcome at 2 years assessed by the Cerebral Performance Category (CPC), and long-term mortality at 6 years. The CPC system was used by the original development studies , 12 and The clinical information was prospectively collected from patient records by the study team. For the calculations of the respective scores, the methodologies of the original publications were strictly applied , 24, 25.In the context of the COMMUNICATE/PROPHETIC study, all patients who had consented to be contacted by the study team were scheduled for a standardised telephone follow-up after 2 years with an assessment of vital status and neurological performance. After the 2-year follow-up period, survival data were obtained by directly contacting either the patient, their relatives, or their general practitioner. If patient contact was not possible, the medical records of the University Hospital Basel (Switzerland) and publicly available death registries were consulted for information concerning vital status. Patients lost to follow-up were censored at the date of the last follow-up. The follow-up time was calculated from the moment of admission to death or censoring date, whichever date came first.According to the original publication of the CAHP score  and a vaχ2-test. Continuous data were checked graphically for normality of the distribution. Continuous, normally distributed variables were compared using analysis of variance (ANOVA) or t-test, and continuous, skewed variables were compared using the Wilcoxon rank-sum test. The scores were calculated according to the original publications [For continuous variables, descriptive statistics such as means, medians, and interquartile ranges were used, and categorical or binary variables were analysed by counts and proportions. Binary and categorical variables between groups were compared using Pearson’s ications , 24, 25.ications . For theDuring the study period, 486 cardiac arrest survivors were admitted to the ICU of the University Hospital Basel, Switzerland. The final analysis included 415 patients, as 38 patients were excluded due to withheld informed consent or screening failure, and 33 patients were lost to follow-up before assessment of vital status at 2 years. Secondary outcome data were available for 80.2% (CPC at 2 years) and 70.4%  of the included patients. Baseline characteristics stratified by the primary endpoint of 2-year survival are presented in Table Of 415 patients, 201 (48.4%) died during the initial hospital stay. Withdrawal of life-sustaining therapy was conducted in 179 of these 201 patients (89%). After 2 years, 241 of 415 patients (58.1%) died. Of the 92 survivors with an assessment of neurological outcome after 2 years, 89 (96.7%) had a good neurological outcome (CPC 1–2), and 3 (3.3%) a poor neurological outcome (CPC 3–4). After 6 years, 241 of 292 patients (82.5%) died. A Kaplan–Meier survival estimate of the total cohort is shown in Additional file χ2 = 19.4, p < 0.001). A graphical comparison of ROC curves for 2-year mortality is shown in Additional file Table The CAHP score showed good calibration for 2-year mortality with a slight overestimation of mortality in the upper two-thirds of the risk spectrum , and two ICU severity-of-illness scores (APACHE II and SAPS II scores) for the prediction of long-term mortality and neurological outcome in a prospective cohort of cardiac arrest survivors followed for up to 8 years. The CAHP score showed the best discriminatory performance for the prediction of 2-year mortality, 6-year mortality, and 2-year neurological outcome. Calibration of the CAHP score was good for 2-year mortality and 2-year neurological outcome. As already demonstrated for the prognostication of short-term outcomes , two nonThe findings of this study are generally in line with previous validation studies evaluating outcomes at hospital discharge or 30 days post-event, where the CAHP score showed a slightly better performance than the OHCA score , 48. In A major and overarching concern in research regarding prognostic factors in post-cardiac-arrest patients is the effect of self-fulfilling prophecy, meaning that the documentation of poor prognosis early in the treatment process per se may lead to a change or withdrawal of care, which again leads to a higher occurrence of poor outcome in this patient group –55. In oThe presented data suggest that the established cardiac arrest-specific scores OHCA and CAHP, which have been thoroughly validated for predicting short-term mortality and neurological outcome in OHCA and IHCA patients, might be suitable for predicting long-term outcomes. Although the OHCA and CAHP scores have originally been intended for the prognostication of outcomes after OHCA, both scores have since been successfully validated in OHCA and IHCA survivors , 56, 57,The OHCA and CAHP score can easily be calculated using openly accessible online calculators, rendering their use easy and straightforward , 59. FurOur study has limitations. First, we did not have complete data for all parameters to calculate the scores and thus had to impute the missing data. Second, due to the study setting, there was a relatively large proportion of loss of follow-up patients resulting in a possible selection bias. This is mainly the case for the secondary outcomes. Assessment of neurological outcome at 2 years of follow-up required a telephone interview, which some patients declined while others could not be contacted by the study team. As a 6-year follow-up was not part of the original study design, a substantial proportion of patients were lost to follow-up. Therefore, the results for 2-year neurological outcome and 6-year mortality have to be interpreted with caution due to a possible selection bias. Third, we only assessed all-cause mortality and thus patients may have died from other unrelated causes. Fourth, the single-centre setting of the study limits the generalisability of the results to other centres, regions or countries. External validation studies evaluating the herein validated scores for long-term outcomes in other populations are necessary to address this issue. However, the relatively large cohort size of the study, the fact that treatment modalities were in line with other Swiss and European medical centres, and the inclusion of unselected cardiac arrest patients indicate a high external validity of the results. Furthermore, analysis and reporting were conducted according to current state-of-the-art methodological guidelines, so that its results can be of the greatest possible use for future research in this field.In our single-centre cohort of cardiac arrest survivors, the OHCA, CAHP, APACHE II, and SAPS II scores showed good performance in early prognostication of long-term mortality at 2 years and acceptable to good performance for the prognostication of 6-year mortality and neurological outcome at 2 years after cardiac arrest. Of the herein validated scoring systems, the CAHP score showed the best discriminatory performance and is a simple-to-use risk-stratification tool available early after cardiac arrest. These scores thus may guide clinicians by stratifying patients according to the risk of poor long-term outcome and may help to support discussions about goals-of-care and the extent of therapeutic effort.Additional file 1: Table S1. Subgroup analysis. Figure S1. Kaplan Meier survival estimate for the entire cohort. Number at risk for the individual time points are reported. Figure S2. Comparison of ROC curves for the primary outcome 2-year mortality. Figure S3. Calibration plot depicting observed vs. expected numbers of primary outcome  per decile of risk as predicted by the CAHP score. Figure S4. Calibration plot depicting observed vs. expected numbers of primary outcome  per decile of risk as predicted by the OHCA score. Figure S5. Calibration plot depicting observed vs. expected numbers of poor neurological outcome at 2 years per decile of risk as predicted by the CAHP score. Figure S6. Calibration plot depicting observed vs. expected numbers of poor neurological outcome at 2 years per decile of risk as predicted by the OHCA score.


Both of these are prepared via remote loading methods, which require multistep procedures. Additionally, their antitumor efficacy is hindered due to the poor drug release from PEGylated liposomes in the tumor microenvironment. In this study, we aimed to develop doxorubicin-loaded lipid-based nanocarriers (LNC-Dox) based on electrostatic interaction using microfluidic technology. The resulting LNC-Dox showed high loading capacity, with a drug-to-lipid ratio (D/L ratio) greater than 0.2, and high efficacy of drug release in an acidic environment. Different lipid compositions were selected based on critical packing parameters and further studied to outline their effects on the physicochemical characteristics of LNC-Dox. Design of experiments was implemented for formulation optimization. The optimized LNC-Dox showed preferred release in acidic environments and better therapeutic efficacy compared to PEGylated liposomal Dox in vivo. Thus, this study provides a feasible approach to efficiently encapsulate doxorubicin into lipid-based nanocarriers fabricated by microfluidic rapid mixing.Doxorubicin (Dox) is a widely known chemotherapeutic drug that has been encapsulated into liposomes for clinical use, such as Doxil Cholesterol was purchased from Merck . Doxorubicin HCl and Doxil® Benchtop from Precision NanoSystems Inc.  was used in this study for the preparation of LNC-Dox. To summarize, LNC-Dox was prepared by mixing neutral lipids , anionic phospholipids , cholesterol, and DSPE-PEG2000 in ethanol with an aqueous solution containing 1 mg/mL solubilized doxorubicin HCl, using a microfluidic system. Production speeds from 1 mL/min up to 12 mL/min were tested. The flow rate ratio (FRR) in microfluidic mixing was controlled in a range from 1.5:1 to 4:1 (aqueous: organic phase). A tangential flow filtration (TFF) system was used immediately for further dialysis and concentration after the preparation of LNC-Dox by microfluidic rapid mixing. The system was run at 28 mL/min with an mPES filter. The final LNC-Dox was stored at 4 °C for further analysis.The NanoAssemblrDoxorubicin was loaded inside liposomes (Lipo-Dox) using an ammonium sulfate gradient. Briefly, the empty liposomes were prepared by mixing 250 mM ammonium sulfate solution and the lipids of HSPC, cholesterol, and DSPE-PEG2000 in ethanol, using a microfluidic system. The total flow rate of the microfluidic system was set at 12 mL/min, and the flow rate ratio of the aqueous and organic phases was 4:1. After the rapid mixing, the liposomes were passed through a size-exclusion chromatograph using a Sephadex G-50 column to replace the buffer with 0.9% NaCl. Then, 1 mg/mL doxorubicin was added to the empty liposomes and incubated at 65 °C for 30 min. The untrapped free-form doxorubicin was removed via size-exclusion chromatography using a Sephadex G-50 column. The final Lipo-Dox was stored at 4 °C for further study.Dynamic light scattering (DLS) was used to measure the hydrodynamic mean diameter and the polydispersity index (PDI) of LNC-Dox. The scattered light intensity of the solution at 173° was determined using an SZ-100 nanoparticle analyzer . Samples were prepared by diluting with deionized water at room temperature. The results were expressed as the mean ± S.D of triplicate samples.®-4, Horiba Jobin Yvon, Edison, NJ, USA).The UV–Visible method was used for the quantification of doxorubicin encapsulated in lipid nanocarriers. Briefly, LNC-Dox was broken down by adding EtOH. Analysis of the amount of doxorubicin in each sample was carried out at 440 nm in a single wavelength mode with a DU 800 UV–Vis spectrophotometer . Standard calibration curves were set up by serial dilution of known amounts of doxorubicin. To determine the cellular amounts of doxorubicin, the fluorescence intensity of doxorubicin was monitored at an excitation/emission wavelength of 470/586 nm with a florescent spectrofluorometer (FluoroMaxThe loading capacity of LNC-Dox was expressed as the drug-to-lipid ratio (D/L ratio), which was calculated as the amount of drug encapsulated inside the LNCs relative to the total amount of lipid. The Bartlett assay was adopted for the quantification of the phospholipid content of LNC-Dox . The amoThe entrapment efficiency (EE) is defined as the ratio of the amount of the drug encapsulated in the LNC relative to the total amount of the drug initially loaded . The amoThe structures of LNC-Dox were examined using an FEI Tecnai G2 F20 TWIN TEM . Samples were prepared for cryo-EM on an FEI Vitrobot by pipetting 4 μL of the reaction mixture containing ~0.5 mM lipids onto a 200-mesh perforated carbon film , blotted for 3 s, and stored in liquid nitrogen until imaging. Images were taken at a defocus value of ~1.8 μm under low-dose exposures (25–30 e/Å2) at 50,000-fold magnification on a FEI Tecnai F20, operating at 200 kV. All experiments were carried out at the Academia Sinica Cryo-EM Facility .® (molecular weight cutoff 100 kDa), immersed in the release medium, at 37 °C and under 100 rpm stirring. Phosphate-buffered saline  and citrate buffer (pH 5.6) were used for mimicking physiological conditions and the acidic tumor microenvironment, respectively [Drug release from liposomes was studied using a dialysis method. Experiments were carried out by placing LNC-Dox samples inside the Float-A-Lyzerectively . DissoluIn order to compare the acidic-triggered release properties of LNC-Dox, the drug release in acidic buffer was normalized against the cumulative drug release in PBS, with the aim to offer a clear view of identifying the extent of acidic-triggered release properties of LNC-Dox ,26. The Briefly, the LNC-Dox was incubated with 20% fetal serum albumin (FBS) at 37 °C for up to 24 h ,28. At t2. The cellular uptake of LNC-Dox and Doxil® was determined by seeding 5 × 105 cells in a 6 cm dish and incubating them at 37 °C in 5% CO2 overnight. LNC-Dox or Doxil® was added to each dish at a concentration of 50 μg/mL and incubated for 2 h. The cells were then washed with PBS twice, lysed with 5x lysis buffer for the determination of protein concentration, and the doxorubicin was extracted with acidic alcohol. The extracted doxorubicin was further quantified by measuring the fluorescence intensity with a spectrofluorometer .C26 murine colon tumor cells were obtained from the Bioresource Collection and Research Center (BCRC) . C26 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin , at 37 °C, in a humidified atmosphere of 5% CO4 cells/mL in 96-well plates and incubated for 24 h. Cells were treated with different concentrations of LNC-Dox and Doxil® for 2 h, and then replaced with drug-free culture medium and further incubated at 37 °C for 24 h. The medium was removed, and the cells were washed with PBS twice, and then incubated with MTT reagent for 1.5 h at 37 °C The amount of MTT formazan produced was analyzed at am absorbance of 570 nm with a DU 800 UV–Vis spectrophotometer . The cells without LNC-Dox treatment were used as controls for 100% viability.The cytotoxicity of LNC-Dox was determined by MTT assay. Briefly, C26 cells were plated at a density of 5 × 105 cells/mL in 6-well plate overnight. Cells were treated with LNC-Dox or Doxil® for 2 h, and then replaced with drug-free culture medium and further incubated at 37 °C for 4 h. Cells were then washed with PBS and lysed in 0.5% (w/v) SDS. The cell lysates were then centrifuged at 800 rpm for 10 min. The supernatant was collected for the quantification of doxorubicin in the cytoplasm, and the pellet was resuspended and lysed with 5x lysis buffer to quantify the amount of doxorubicin in the nucleus. The accumulation of doxorubicin in the nucleus and cytoplasm was presented as the percentage associated with the total amount of internalized doxorubicin. Doxorubicin accumulated in cell nuclei was determined as described in the report of Li et al. . BrieflyA0 is a constant, A1 to A9 are the regression coefficients, and X1, X2, and X3 are the independent factors. Analysis of variance (ANOVA) was used to examine the correctness and reliability of the models, including the coefficient of determination (R2), R-squared (adjusted), R-squared (predicted), and lack of fit . Moreover, 3D response surface graphs were generated to depict the influences of variable factors. The best possible formulation of LNC-Dox was selected and evaluated based on the results of RSM to achieve the optimal critical attributes of LNC-Dox. Meanwhile, the error percentages between the experimental and predicted values of critical attributes were examined for the accuracy and conformity of the models.To optimize the main effects, quadratic effects, and interaction effects of the formulation ingredients and process parameters, RSM combined with the BBD scheme was used in this study. BBD with three three-level factors—including the amount of DOPA, amount of DOPE, and FRR—was applied. Design-Expert 12 software was used to generate the 16 experimental runs, including 3 center-point trials, for further evaluation of the optimized LNC-Dox. The observed responses included particle size, polydispersity index (PDI), entrapment efficiency (EE%), drug-to-lipid ratio (D/L ratio), stability, and normalized drug release. The results obtained for each response were fitted to a second-order polynomial model used in the RSM, which is explained by Equation (5): 5 cells) without a matrix were implanted into the right-side backs of mice (6–8 weeks of age) by subcutaneous injection. Mice (n = 5/group) were randomly arranged and as assigned to the control and the experimental groups. The free form-doxorubicin, liposomal Dox, and LNC-Dox were diluted with 0.9% NaCl. The drug concentration for intravenous injection was 5 mg/kg via the tail vein when the tumor grew to 100 mm3 in volume. In the control group, 0.9% NaCl was given. The tumor size and body weight of the mice were monitored every 2 days. Tumor volume of 2500 mm3 was considered as the end point, upon which the mice would be euthanatized by CO2 inhalation.The in vivo antitumor efficacy against C26 murine colon tumor cells was determined in BALB/c mice obtained from the National Laboratory Animal Center . The protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Taiwan University . C26 cells  and flow rate ratio (FRR)—have significant effects on the physicochemical properties of nanocarrier formation ,32. TherII phase or inverted micelles, in the mixture of doxorubicin and anionic lipid EPG. As mentioned above, EPG was used in the present formula to increase the association between doxorubicin and the carriers. Therefore, we further examined the possible differences in the characteristics and morphological structure of Dox-loaded nanocarriers prepared with or without EPG. The results of dynamic light scattering and D/L ratio are shown in Previous studies have shown that RNA-encapsulated lipid nanoparticles exhibit an electron-dense core, which might consist of reverse micelles or inverse hexagonal structures formed by the electrostatic interaction between anionic nucleic acids and ionizable lipids . As showp-value, a quadratic model was suggested for studying the stability, while the linear models were proposed for analyzing particle size, D/L ratio, and normalized drug release. Meanwhile, a 2F1 model was carried out to examine the entrapment efficiency. As shown in As shown in As shown in the contour plot of p-values of the other polynomial models were insignificant. 2 for the RSM model, implying that the overall mean might be a better predictor of the response. In addition, the changes in the lipid composition and the FRR had insignificant effects on the PDI value, as shown in As shown in the Based on the function of DOPA%, DOPE%, and FRR , the 3D During microfluidic processing, the FRR could influence the solution’s polarity, which might have affected the drug-loading capacity in forming the nanocarriers. Therefore, we addressed the impact of FRR on the EE and D/L ratio of LNC-Dox; as shown in Additionally, both factors—DOPA% and DOPE%—were found to be significant in explaining the response of normalized drug release, as shown in the + in the acidic environment, the tight complexation was not easily reversed, resulting in a higher error percentage between the actual and predicted values of the normalized drug release. However, the normalized drug release was still elevated to 3.15 (Based on the above optimization studies, RSM with a BBD scheme provided the best possible formulation, which achieved the desired responses, including the maximum stability, EE%, D/L ratio, and normalized drug release. The best possible formulation for LNC-Dox was composed of DOPA:EPG:DOPE:HSPC:cholesterol:DSPE-PEG2000 at a molar ratio of 37.44:9.36:0.47:8.93:41.9:1.9, and the FRR was set to 5:1. The prepared LNC-Dox was tested, and the experimental responses were further compared to the predicted values to validate the accuracy. As shown in  to 3.15  compared to 3.15 b. In fac to 3.15 a, as com to 3.15 a for the to 3.15 b. These ®. However, there was no significant difference in the cellular uptake between these two different doxorubicin-loaded lipid nanocarriers (®. These results indicate that the preferred release of LNC-Dox in acidic environments could enhance the intracellular drug release and enter the nucleus to exert better therapeutic efficacy.To further evaluate the therapeutic efficacy of optimized LNC-Dox composed of EPG, DOPA, cholesterol, HSPC, DOPE, and DSPE-mPEG2000, we first examined its in vitro cytotoxicity against murine C26 tumor cells. As shown in carriers b. As rep®. In fact, the overall survival rate was also significantly improved in mice treated with the optimized LNC-Dox compared to that those treated with Doxil® (middle panel of ® in terms of suppressing tumor growth. Doxorubicin encapsulated in PEGylated liposomes is released both in the tumor’s extracellular fluid, and intracellularly via the endosomal escape process [The in vivo therapeutic efficacy of optimized LNC-Dox was further examined in BALB/c mice bearing C26 tumors. As shown in the left panel of  process . However process . As show®. There are two advantages of using this LNC-Dox for tumor treatment: First, doxorubicin encapsulated in this LNC delivery system had a high D/L ratio, which could offer a higher therapeutic dose without excessive use of lipids for clinical development. Second, the preferred release in acidic environments could increase the amounts of bioavailable drug for tumor treatment. Therefore, the strategy used in the present formulation’s development, combined with the bottom-up microfluidic technique, showed its potential in improving drug-loading capacity and simplifying the manufacturing processes for the translational development of lipid-based nanocarrier drugs. In this study, Dox-loaded lipid-based nanocarriers composed of neutral and anionic phospholipids were developed and fabricated by microfluidic rapid mixing. The optimized LNC-Dox showed better tumor regression and overall survival rates compared to the commercial drug DoxilThe work reported in this manuscript has a patent pending under the title of composition and method of preparation for lipid formulations comprising charged lipids.


Left atrial appendage (LAA) is a common source of thrombi in patients with atrial fibrillation (AF). The effect on left atrial (LA) function of the Totally Thoracoscopic (TT)-LAA exclusion with epicardial clip is currently unknown. This study aims at evaluating the effect of TT-LAA exclusion on LA function.Standalone TT-LAA exclusion with the clip device was performed in 26 patients with AF and contraindication to oral anticoagulation (OAC). A 3D CT scan, trans-esophageal echocardiography, spirometry and cerebrovascular doppler ultrasound were performed preoperatively. Clip positioning and LAA exclusion were guided and confirmed by intraoperative trans-esophageal echo. To evaluate LA function, standard transthoracic echocardiography and 2D strain of LA were performed before surgery, at discharge and at 3-month follow-up.2DS2-VASc and HASBLED scores were 4.6 and 2.4 respectively. There were no major complications during the procedure. At median follow-up of 10.3 months, 1 (3.8%) non-cardiovascular death, 1 (3.8%) stroke and 4 (15.4%) cardiovascular hospitalizations occurred. At 2D strain of LA, the reservoir function decreased significantly at discharge, compared to baseline and recovered at 3-months follow-up. Furthermore, NT-proBNP increased significantly after the procedure with a return to baseline after 3 months. Changes in E/A were persistent at 3 months.The mean CHAOur data in a small cohort suggest that TT-LAA exclusion with epicardial clip can be a safe procedure with regards to the atrial function. The LAA amputation impairs the reservoir LA function on the short term, that recovers over time. The surThis study aims at evaluating the effect of totally thoracoscopic (TT)-LAA exclusion on the LA function, evaluated with 2D STE.This observational and retrospective study enrolled patients with AF at high risk for ischemic stroke and at high risk of life-threatening bleeding on OAC or APT or with a contraindication to long-term OAC. All patients underwent TT-LAA exclusion in the period between March 2020 and June 2021 at S. Chiara Hospital, Trento, Italy.Inclusion criteria were: 1) AF defined following current guidelines  AF defin.Final decision on inclusion in the study was taken by the “AF Heart Team,” including a cardiac surgeon, a cardiologist, neurologist/neurosurgeon and referring physician following current guidelines on LAA exclusion .All patients underwent preoperative computed tomography (CT) with 3-dimensional reconstruction, transthoracic and transesophageal echocardiography (TEE) to rule out thrombi in the LAA and to exclude other cardiac surgery indications for structural or functional heart diseases. Spirometry and bilateral carotid ultrasound doppler were also performed during the preoperative work out. Clinical history and laboratory data were collected and analyzed. Patient provided written informed consent to the procedure. The study complied with the Declaration of Helsinki as revised in 2013; the ethic committee approved the study.2, visualization of the intrathoracic anatomy, and freeing of adhesions, the opening of the pericardium was performed. The LAA was mobilized and the base measured with a dedicated sizer to select the device size. The AtriClip PRO2  device was positioned using the dedicated deployment device and deployed under TEE guidance and camera visualization.All patients were treated using the video assisted thoracoscopic LAA exclusion approach with Atriclip PRO2 device . The procedure has been previously described in details . BrieflyEchocardiographic analysis was performed in all patients by experienced cardiologists, a specific protocol was compiled, and the echocardiographic measurements were obtained following current guidelines . StandarTwo-dimensional speckle tracking echocardiography was performed with a standard protocol following current guidelines . The apiPeriprocedural adverse events were registered. Thoracoscopic access was evaluated after 10 days from the procedure. Clinical evaluations included laboratory work-out at pre-discharge and after 3 months and physical examination. A protocol echocardiogram was performed at baseline (pre-surgery), at pre-discharge (after surgery) and at 3 months. At 3 months follow-up, a cardiac synchronized CT scan or TEE were also performed to measure the size of the residual stump, if any and to assess the efficacy of LAA exclusion. A satisfactory outcome was considered as a residual stump < 1 cm . The priT-test was used to compare numerical normal variables, and Wilcoxon test for non-parametric variables. The categorical variables were compared by Chi-squared test or Fisher’s exact test and described as frequencies and percentages. A p-value < 0.05 was considered significant for all tests. The analysis was performed using R software version 3.6.2 .Descriptive statistics are reported as medians and interquartile range (IQR) for non-normally distributed continuous variables or mean ± standard deviation for normally distributed continuous variables. 2DS2-VASc scores were 2.4 ± 0.6 and 4.6 ± 1.1 respectively. Permanent AF was present in 8 patients (30.8%). The indication for LAA exclusion was the following: history of cerebral hemorrhage (n = 10), diffuse gastrointestinal hemorrhage requiring transfusions (n = 4), clinical scenario of high bleeding risk (n = 4), refractory anemia (n = 3), other bleeding scenarios (n = 2), refractory LAA thrombosis (n = 1) and recurrent stroke despite different OAC therapies (n = 2). Baseline characteristics are summarized in The study enrolled 26 consecutive patients . The mean HAS-BLED and CHAA total of 26 patients underwent thoracoscopic LAA exclusion. Mean operation time (skin-to-skin) was 69.2 ± 18.5 min. No deaths procedure-related or pulmonary morbidity were observed. No patient required conversion to mini thoracotomy. Intraoperative TEE showed complete LAA exclusion with minimal residual stump (<1 cm) in all cases. Following the procedure, no patients were prescribed OAC, APT or heparin. There were no major complications during and after the procedure.p < 0.001); after 3 months there was no differences in NT-proBNP value compared with baseline (p = 0.17)  . There w = 0.17) .p = 0.028) with a recovery at 3-months  . At pre-discharge and at 3-months follow-up echocardiography, the LA volume indexed to BSA was unchanged compared with baseline measurements . There was no significant difference in LV EDV, LVEF and GLS of LV  . When co = 0.55) . Of noteLS of LV . The resFollow up was completed and available for all 26 patients. At a median follow-up of 10.3 ± 4.7 months, no patients were on OAC, APT or heparin therapy. One (3.8%) non-cardiovascular death, 1 (3.8%) stroke and 4 (15.4%) cardiovascular hospitalizations occurred at long-term follow up. Evaluation by TEE or CT after 3 months showed stable and appropriate device position with LAA stump < 1 cm in all patients.The main findings of this study can be summarized as follows: (1) The amputation of LAA significantly impairs LA reservoir function after the procedure, although this function recovers after 3 months. (2) TT-LAA exclusion results in a change in E/A that is persistent at 3 months follow-up.The LA function consists of three components, namely: conduit, reservoir and pump. It is the result of a complex interplay between LV systolic and diastolic function, circulating blood volume and LAA function .In the current study a transient impairment of LA reservoir function was observed after LAA exclusion; different mechanisms might contribute to this finding.The sudden volume reduction of the LA after the procedure may affect LA distension, whereas its recovery might be explained with volume recovery or LA remodeling over time.Changes in LA function could also be secondary to an altered neuro-humoral homeostasis expressed by changes in both atrial natriuretic peptide (ANP) and NT-proBNP. LAA endovascular occlusion is associated with an increase in ANP levels ; ANP is Previous studies with percutaneous LAA closure, were characterized by heterogeneous results; different groups showed no changes in LA function after the procedure –19. A liThe recovery of reservoir strain of LA after 3 months is consistent with previous studies with 2D STE , 23; it Pulsed wave measures, in particular E/A remained increased at 3 months follow-up in the current study. In the pulsed wave analysis of transmitral flow, E wave represents the early fast diastolic filling and it is a measure of LA reservoir function. Its increase, with a consequent increase of E/A has been reported in previous studies ; it coulThe main limitation of the study is that it is retrospective. The included number of patients was relatively small, due to strict inclusion criteria. Limitations also included referral bias, being the center specialized in TT treatment of AF and TT-LAA exclusion. The reported changes in reservoir function might depend also on the appendage volume, which may differ among individuals. Data on LAA volume are lacking in all the published studies, and in the present one. In patients with permanent AF, A wave was not measured.Our data in a small cohort suggest that TT-LAA exclusion with epicardial clip can be a safe procedure with regards to the atrial function. The LAA amputation impairs the reservoir LA function on the short term, that recovers over time.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Santa Chiara Hospital Ethics Committee. The patients/participants provided their written informed consent to participate in this study.MMa, LP, and CA: conception and design of the work. MMa, LP, SB, FT, GD’O, DF, and ES: substantial contributions to the acquisition of data for the work. MMa and LP: substantial contributions to the analysis of data for the work and drafting the work. AG, RB, MMe, GC, and CA: substantial contributions to the interpretation of data for the work. FG, SQ, CM, AG, RB, MMe, GC, and CA: revising the draft of the work critically for important intellectual content. MMa, LP, SB, FT, GD’O, DF, ES, FG, SQ, CM, AG, RB, MMe, GC, and CA: final approval of the version to be published and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors contributed to the article and approved the submitted version.


The MCM-T-H-SPME showed excellent reproducibility (RSD < 9.8%) and fast adsorption kinetics (30 min) and desorption kinetics (5 min) for derivatized BAs under optimal conditions. In summary, the MCM-T-H-SPME Arrow based method was employed for accurate monitoring of the variations of BAs in pork and fish, and good results were achieved.In this study, a functionalized mesoporous silica-coated solid-phase microextraction (SPME) Arrow system was developed for the enrichment of six biogenic amines (BAs) from pork and fish samples before gas chromatographic separation with a mass spectrometer as a detector. MCM-41 was utilized as the substrate material and thereby functionalized by titanate and sodium dodecyl sulfate to adjust its surface acidity and hydrophobicity, respectively. The functionalized MCM-41 (named as MCM-T-H) was coated on a bare SPME Arrow using the dipping method and polyacrylonitrile was used as the adhesive. The extraction capacity and selectivity of the MCM-T-H-SPME Arrow for six kinds of derivatized BAs were studied and compared with commercial SPME Arrows. Experimental parameters, e.g., sample volume, derivatization reagent amount, extraction time, and desorption time, which have a dramatic effect on SPME Arrow pretreatment, were optimized. Acidity enhanced MCM-T-H coating showed a much higher affinity to derivatized BAs compared to a commercial SPME Arrow in terms of extraction capacity. In addition, hydrophobicity modification significantly reduced the interference of water molecules on the interaction between MCM-T-H and the derivatized BAs. The MCM-T-H-SPME Arrow showed efficient separation and enrichment capacity for derivatized BAs from complex matrices and therefore, the sample pretreatment time was saved. According to the experimental results, the optimal condition was to add 10 μL derivatization reagent to a 10 mL sample and maintain an agitation speed of 1250 r min Their chemical structures contain one or more amino groups , and a large pore volume (0.5–3.0 cm3 g−1). The aperture distribution is narrow, and the material with a diameter of 20–300 Å can be made by changing the manufacturing process [MCM-41 is a type of mesoporous silicon material , which h process . However process . On the Sodium dodecyl sulfate (SDS) is a kind of surfactant that is soluble in water and its ester linkage can be hydrolyzed when heated. This creates a hydrophobic surface for the particles and improves the compatibility between the particles and the analytes . NikosokThere are many classic analytical methods for the determination of BAs such as thin-layer chromatography , ion chrThe purpose of this study was to prepare the functionalized mesoporous silica MCM-T-H with excellent adsorption capacity and fast kinetic extraction to derivatized BAs from different sample matrices. MCM-T-H was synthesized based on mesoporous MCM-41 and was then utilized as the coating material of the SPME Arrow using a straightforward dipping strategy. MCM-T-H series materials were fully characterized to investigate the extraction mechanism of MCM-T-H to derivatized BAs. The preparation parameters that affected the MCM-T-H coating extraction performance were also studied. After optimizing the SPME parameters, the applicability of MCM-T-H SPME Arrows was further evaluated by exploiting them for the detection of BAs in pork and mackerel samples.4OH) were purchased from Macklin . Anhydrous toluene and hydrochloric acid (HCl) were from Luoyang Haohua Chemical Reagent Co., Ltd. . The ultrapure water used in the study was from a ultrapure water system  with a resistivity of 18.25 MΩ·cm. The headspace bottle and PTFE/silicone septum screw cap were from Merck KGaA . The commercial SPME Arrow with the Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) coating and the bare SPME Arrow were purchased from CTC Analytics . The stock solutions containing six BAs were prepared by dissolving them in HCl at 0.1 mol L−1 and stored at 4 °C. A series of working solutions were obtained by diluting the stock solutions properly with ultrapure water. Then the pH of the working solutions was adjusted to 11 with 2 mol L−1 NaOH. For each SPME Arrow extraction, 10 mL of the working solution was added to the sample vial for the subsequent SPME Arrow extraction.Phenylethylamine (PHE), putrescine (PUT), cadaverine (CAD), histamine (HIS), tryptamine (TRY), tyramine (TYR), cetyltrimethylammonium bromide (CTAB), TTIP, SDS, tetraethyl silicate (TEOS), ethanol, polyacrylonitrile (PAN), isobutyl chloroformate (IBCF), N-N dimethylformamide (DMF), trichloroacetic acid (TCA), sodium hydroxide (NaOH), and ammonium hydroxide solution (NHThe detection of BAs was conducted by gas chromatography–mass spectrometry (GC–MS) . The chromatographic column was a HP-5 MS . Surface groups of the materials were analyzed by a fourier transform infrared spectrometer (FTIR) . Powder X-ray diffraction (XRD) patterns were recorded on a Bruker D8 Advance . Transmission electron microscopy (TEM)  was used to observe the interior structure of materials. The thermal stability of the materials was evaluated by a thermal gravimetric analyzer (TGA) . A Brunauer–Emmett–Teller (BET)  method was used to evaluate the specific surface area and pore size of the materials. An energy dispersive X-ray (EDX) with a JXA-8230  provided a chemical components analysis of the samples to determine their chemical composition when utilized it in combination with scanning electron microscopy (SEM) .4OH and stirred for 5 min. Then, 10 mL of TEOS was added slowly into the solution and stirred at room temperature for 2 h. The precipitation was filtered, dried, and finally calcined at 550 °C for 6 h in the air to remove templates.MCM-41 synthesis referred to by Song et al. in . BrieflySurface grafting techniques were used to modify the TTIP on MCM-41 and the subsequent material was noted as MCM-T. One gram of MCM-41, 20 mL of anhydrous toluene and 50 µL of TTIP were added in a three-neck flask . After sMCM-T functionalization with SDS was referred to by Bing et al. in . FunctioAs shown in −1, held at 100 °C for 1.2 min, increased to 160 °C at 10 °C min−1, and finally ramped to 280 °C at 25 °C min−1, and held for 12 min [−1. The temperature of the transfer line, ion source, and analyzer were 280, 230, and 150 °C, respectively. Derivatized BAs were ionized using the electron ionization (EI) mode and scanned using the full scanning mode.First, 10 mL of the working solutions  was added into a 20 mL headspace vial and mixed with 10 µL of derivatization reagent IBCF. Then, the sample vial was sealed with a PTFE/silicone septum screwcap and placed on the magnetic stirrer for stirring. The direct immersion technique was used for the sampling with the SPME Arrow. The MCM-T-H-SPME Arrow punctured the septum and the coating part was exposed in the liquid sample for derivatized BAs extraction. After that, the SPME Arrow was inserted into the GC inlet for thermal desorption and GC–MS analysis. The desorption temperature was 250 °C and the desorption time was 5 min. The oven was programmed according to the following temperature program: the temperature was increased from 50 to 100 °C at 50 °C minr 12 min . The tot–1 NaOH. For the SPME Arrow extraction, 10 mL of the pretreated real sample was added into a 20 mL headspace vial and mixed with 10 µL of derivatization reagent IBCF for the subsequent SPME procedures.Pork and mackerel were used as the template food samples and they were purchased from local supermarkets in Ningbo. Pork and mackerel were sampled at the same time over seven consecutive days. First, 10 g of the sample was placed in a centrifugal tube, followed by adding 20 mL of 5% TCA, and the mixture was homogenized for 2 min. The supernatant was collected after centrifugation. The above operations were repeated twice then the supernatants were merged into a 50 mL volumetric flask and the final volume was fixed to 50 mL using 5% TCA. Finally, the pretreated samples were stored at 4 °C. The pH of the working solution was adjusted to 11 by using 2 mol LAll materials  were characterized by FT-IR, XRD, BET and SEM . The opt−1 were assigned to the stretching and bending vibration modes of the hydroxyl groups of physically adsorbed water molecules [−1 could be ascribed to the presence of a Si-OH group, which was the main characteristic group of MCM-41, which also existed in MCM-T and MCM-T-H. Two peaks at 1088 cm–1 and 802 cm–1 were ascribed to asymmetric and symmetric stretching vibrations of Si-O-Si [−1, which was usually attributed to the presence of the Si-O stretching vibration [−1 was due to stretching vibration caused by the bonding of SiO2 tetrahedron with the Ti atom through the Ti-O-Si bond [–1 is slightly smaller than that of MCM-T and MCM-T-H, which indicated that TTIP had been successfully modified on the surface of MCM-41. Both SDS and MCM-T-H had absorption peaks at 2923 and 2852 cm–1, which were the stretching vibration peak of -CH2 [olecules . The obs Si-O-Si . The FTIibration . However-Si bond . The inc of -CH2 . They diTypical small-angle XRD patterns of three materials were shown in Three materials show a type IV adsorption isotherm, indicating the mesoporous nature of the materials d. The adIn order to further study the surface morphology characteristics of the three materials, SEM and EDS characterizations were carried out . The par2 g–1; pore diameter: 2.37 nm; total pore volume: 0.73 cm3 g–1) showed the highest extraction capacity to derivatized BAs. Therefore, 0.25% of CTAB was selected as the optimal additive amount to synthesize MCM-41.MCM-41 is a kind of amorphous mesoporous silica material with a unique hexahedral structure [–1  was partly due to the structural collapse of MCM-41 upon incorporation with denser Ti species [Incorporation of Ti into mesoporous silica generates a large number of Lewis and Brønsted acid sites, which further improves the acidity of the material and thus enhances the adsorption affinity to BAs . MCM-T s–1 b. Comparectively ). Pore s species .2 g–1; pore diameter: 1.87 nm; total pore volume: 0.47 cm3 g–1)  . SurfaceDuring the preparation of the SPME Arrow, the increase of coating cycles could raise up the thickness of the coating and therefore increase its extraction capacity. The bare SPME Arrow had an outer diameter of around 320 μm . With anThe MCM-T-H-SPME Arrow was compared with the commercial DVB/CAR/PDMS-SPME Arrow, and the result is shown in −1) was used. As predicted, the MCM-T-H coating showed the best extraction efficiency when the sample volume was 5 mL  since a BAs–IBCF reaction was easier to carry out in a basic solution. IBCF is acidic, therefore, an excess of IBCF significantly reduced the pH, which hindered the derivative reaction process. However, too little IBCF could not guarantee the completion of the derivatization reaction. After a compromising consideration and according to the optimization results , 0.9–5.12 mg kg–1 (PUT), 0.10–9.48 mg kg–1 (CAD), to 0.43–3.25 mg kg–1 (TYR), respectively. The concentrations of PHE, PUT, CAD, HIS, TRY, and TYR in mackerel were varied in the range of 5.2–234 mg kg–1 (PHE), 4.5–457 mg kg–1 (PUT), 0.5–585 mg kg–1 (CAD), 6.8–657 mg kg–1 (HIS), 0.6–46.6 mg kg–1 (TRY), and 0.3–696 mg kg–1 (TYR), respectively. After three days of storage, the mackerel has spoiled and was inedible while the BAs were still at a quite low concentration in pork which is probably due to the higher protein, water, and bacterial contents in mackerel. BA recoveries from pork and mackerel were 78.5–123% and 74.6–118%, respectively. Obviously, some recovery results were higher than 100% which could be for two reasons. First, the operation error and instrumental error leads to deviation of recoveries. Second, the homogeneity of the sample is also an important factor which affects the recovery result. The spiked levels of several BAs were higher than the LOQ values because we spiked the BAs based on their concentrations in the pork and fish samples. For example, the CAD concentration in the mackerel on day three was 352 ± 22 mg kg–1, therefore 350 mg kg–1 of CAD was spiked into the fish sample to evaluate the recovery of CAD using our developed method. On the other hand, since some BA concentrations exceeded the linear range of our method, we processed a preliminary experiment to estimate the BAs’ concentration and then we diluted the extraction solutions of pork and fish samples using a basic solution (pH = 11). Overall, those results indicated that the MCM-T-H coating was stable and reliable to enrich derivatized BAs efficiently from complicated food matrices. In addition, the developed MCM-T-H-SPME Arrow GC–MS method is applicable to precisely monitor BAs variation with satisfactory recovery.The applicability of our developed method was validated by monitoring BA concentrations in pork and mackerel, which were stored at room temperature (22 ± 2 °C) for seven days in a row. Over those days, variations of BAs in pork and mackerel from each day were analyzed with triplicate measurements. In addition, pork and mackerel samples on the third and sixth storage days were used for a recovery test to evaluate the precision and accuracy of this method. Monitoring results are summarized in Overall, a SPME Arrow GC–MS method based on the hydrophobic mesoporous silica was successfully developed for qualitative and quantitative analysis of six BAs in pork and fish. The mesoporous MCM-41 was grafted with titanium, and thereby a large number of Lewis and Brønsted acid sites were generated, which further improved its acidity, and the extraction efficiency of the acidified MCM-41 to derivatized BAs was obviously increased. SDS was further modified on MCM-T to increase its hydrophobicity so that the interference of water molecules could be avoided as much as possible. According to the results, the hydrophobic MCM-T-H coating has a high-extraction affinity to derivatized BAs rather than water molecules. The MCM-41 series materials were comprehensively characterized by multiple characterization techniques and the selective extraction mechanism of MCM-T-H to derivatized BAs was studied. The SPME Arrow GC–MS method developed showed excellent sensitivity, selectivity, and reproducibility and it also showed good applicability for the detection of six BAs from pork and fish samples.n = 3), and the coating was stable up to 50 extraction and desorption cycles. Compared to the commercial SPME fiber, the MCM-T-H-SPME Arrow has the advantages of being low cost and robust. In addition, the MCM-T-H-SPME Arrow has a better extraction performance for the six derivatized BAs than the commercial DVB/CAR/PDMS-SPME Arrow. The MCM-T-H-SPME Arrow GC–MS method developed here can accurately monitor the variation of BAs in pork and fish samples with good recovery. Therefore, this method provides a promising alternative to conventional BA detection methods and extends the application of the SPME Arrow to food safety. Moreover, the material is simple to synthesize with a low cost, which will greatly reduce labor and other costs in the traditional food monitoring field.To summarize, hydrophobic MCM-T-H was prepared and used as the coating material of an SPME Arrow. The average thickness of the MCM-T-H coating was 4 µm with RSD less than 9.8% (


To evaluate the performance of single-echo Dixon water-fat imaging and computed tomography (CT)–like imaging based on a single ultrashort echo time (sUTE) MR sequence for imaging of vertebral fractures as well as degenerative bone changes of the spine in comparison to conventional CT and MR sequences.Thirty patients with suspected acute vertebral fractures were examined using a 3-T MRI, including an sUTE sequence as well as short-tau inversion recovery (STIR) and T1-weighted sequences. During postprocessing, water-fat separation was performed by solving the smoothness-constrained inverse water-fat problem based on a single-complex UTE image. By removing the unwanted low-frequency phase terms, additional MR-based susceptibility-weighted-like (SW-like) images with CT-like contrast were created. Two radiologists evaluated semi-quantitative and quantitative features of fractures and degenerative changes independently and separately on CT and MR images.κ 0.90 ; AO/Magerl: κ 0.75 ). Overall inter-reader agreement for water-fat-separated UTE images and SW-like images was substantial to almost perfect.In total, all 58 fractures were accurately detected of whom 24 were correctly classified as acute fractures with an edema detected on the water-fat-separated UTE images, using STIR and T1w sequences as standard of reference. For the morphological assessment of fractures and degenerative changes, the overall agreement between SW-like images and CT was substantial to excellent (e.g., Genant: Detection and assessment of vertebral fractures and degenerative bone changes of the spine were feasible and accurate using water-fat-separated images as well as SW-like images, both derived from the same sUTE-Dixon sequence.• The detection of acute vertebral fractures was feasible using water-fat-separated images and CT-like images reconstructed from one sUTE sequence.• Assessment of the vertebral fractures using SW-like images with CT-like contrast was found to be comparable to conventional CT.• sUTE imaging of the spine can help reduce examination times and radiation exposure. Spine pathologies and degenerative changes are associated with back pain and disability and are among the greatest risk factors for morbidity and mortality worldwide , 2. For 2* relaxation times [2* decay of water signal into account [2, again requiring the acquisition of two complex signals to solve the background phase terms in postprocessing which prolonged the scan time [For the assessment of the fracture age, STIR- and T1-weighted sequences are usually acquired to identify edema-equivalent bone marrow changes, which are considered to be highly specific for bone marrow edema after an acute trauma , 13. Recon times –18. Howeon times . However account . Single- account , 20, 21.can time .The purpose of this study was to evaluate the diagnostic value of a newly developed method  that simThe study was approved by our institutional review board . Informed consent was obtained from all study participants prior to inclusion. Participants which were admitted to the emergency unit were recruited from November 2018 until September 2019. In total, 30 patients  with suspected acute thoracolumbar vertebral fracture were included into the study. All patients received a CT scan of the spine as part of the routine clinical diagnostic work flow and received a MRI within 3 days.Each patient received a CT scan of the thoracolumbar spine using either a Philips IQon Spectral CT scanner or a Siemens Somatom Definition AS+ scanner. Clinical scan parameters were set according to the clinical routine: collimation, 0.6 mm; pixel spacing, 0.4/0.3 mm; pitch factor, 0.8/0.9; tube voltage (peak), 120 kV; and modulated tube current, 102–132 mA. Images were acquired in axial orientation and reformatted in sagittal and coronal orientation using a bone-specific convolution kernel .and a sagittal STIR sequence. To measure the signal of tissues with short T2* values, a 3D UTE stack-of-stars sequence was employed [3, voxel size (acquired) 0.45 × 0.45 × 3 mm3, voxel size (reconstructed) 0.28 × 0.28 × 0.75 mm3 and acquisition time 6.3 ± 0.23 min, ramp length 0.08 ms, max. gradient strength 15.04 mT/m, sampling dwell time 3.12 μs with 568 samples, acquisition window 1.77 ms, 945 number of spokes, with radial percentage of 85%, partial Fourier with a factor of 0.6 in slice direction. Image reconstruction was performed offline and gradient imperfections were corrected by means of a gradient impulse response function [For MR imaging, a standard spine protocol was used including a sagittal T1-weighted sequence with turbo spin echo (T1w TSE) employed , 24 withB0 inhomogeneities, eddy currents, signal delays in the receiver chains, and the B1 transmit/receive phase. Due to the smoothly and slowly varying nature of these unwanted phase terms, an iterative and smoothness constrained approach was used as done previously [For postprocessing of the single-echo UTE images, sUTE-Dixon water-fat separation was performed. The algorithm solves the water and fat inverse problem while simultaneously removing unwanted phase terms in the UTE phase. The unwanted phase terms were mainly composed of phase due to eviously . The neweviously .n = 1–5 times multiplied with the magnitude of the original UTE image to achieve an improved contrast S′n. In order to achieve a CT-like contrast with high “bone-like” signal, the SW-like images were inverted.SW-like images were created by using the corrected UTE phase from the sUTE-Dixon processing. Based on the corrected UTE phase, phase masks were generated and scaled between 0 and 1 to increase bone contrast. The phase masks were then CT and MR images were read by two radiologists . The images were read individually and independently in a random order and blinded to clinical information and any other imaging data. The different MR sequences and CT images were read with at least 8 weeks in between readings, respectively. For intra-reader reproducibility, 10 patients were assessed once again after 8 weeks by both radiologists. Image analyses were performed on a PACS work station certified for clinical use .The images were assessed for the presence and exact location of vertebral fractures. For the evaluation of the fracture age, STIR and water-separated sUTE images were analyzed for the presence of edema-like bone marrow signal. T1-weighted images and fat-separated sUTE images were evaluated for a corresponding signal drop. Additionally, UTE-based SW-like images and CT were assessed for the visibility of fracture lines and contrast of the vertebrae using a 5-point Likert scale . Morphological features of the spine were analyzed and graded according to previously described clinical scores . Fracturα) of 0.05 was used. Agreement of ordinal scaled parameters was assessed using weighted Cohen’s κ [t-tests (for numeric variables) and McNemar’s tests .The data were analyzed using IBM SPSS Statistics for Windows, version 27.0 (IBM Corp.). All statistical tests were performed two-sided and a level of significance , with a slight difference depending on the phase masks applied  of the fractures were classified as AO3.κ 1.00 ) ; Genant classification, κ 0.89 ). The overall agreement regarding the evaluation of the fracture age was excellent, κ 0.77 , and for degenerative changes substantial to almost perfect, κ 0.90 ) Figs. , using t and 2: )N = 58, κ 1.00 (95% CI 1.00–1.00) for both readers, Figs. All vertebral fractures were accurately detected using SW-like images with CT-like contrast by both readers ) to almost perfect ).AO classification of the fractures was graded identical for SW-like images with 5 phase masks applied by both readers compared to CT (readers 1 and 2: κ 0.62 (95% CI 0.35–0.84) for the contrast of the vertebrae to perfect ) for classification of the fracture age and edema-like signal detection (Table κ 0.82 (95% CI 0.59–1.00) for the fracture age to κ 1.00 (95% CI 1.00–1.00) for the fracture classification (AO/Genant) and detection of osteophytes (Table Inter-reader agreement for water- and fat-separated sUTE images was excellent ranging from κ 0.71 to 1.00) for both readers  for both readers).For intra-reader reliability, both readers assessed the images of 10 patients after at least 8 weeks separately, independently and blinded to all clinical information. The intra-reader agreement was overall substantial to almost perfect  as well as in most cases a CT for the evaluation of the fracture itself. Using the proposed image reconstructions of the UTE sequence, both SW-like images with CT-like contrast and water-fat-separated images can be reconstructed from one single UTE sequence, which consequently may save scan time, since in theory no additional sequences or examinations would be needed. The reconstructions are performed on a separate server.Yet, compared to UTE imaging, ligaments, calcifications, and other tissues with short TThere are some limitations to this study. Although the radial acquisition of the images made the scan less motion sensitive, the acquisition time of approximately 6.3 min itself creates a certain susceptibility to motion artifacts. Furthermore, the UTE was susceptible to slight fat blurring due to the radial k-space trajectory which depends on the readout bandwidth and the in-plane resolution of the scan protocol. In some cases, a slight hyperintense signal was seen in the water-separated sUTE images adjacent to the fractures which was presumably caused by the vertebral venous plexus and was misinterpreted in two cases as an acute fracture. The signal of the presumed vertebral venous plexus usually appeared to be weak hyperintense with a lower signal than the signal of an acute bone marrow edema caused by a fracture. Yet, when assessed simultaneously with the SW-like images, the vertebral venous plexus can be differentiated in the vast majority of the cases from an acute fracture due to the weaker signal hyperintensity compared to edema and the missing fracture morphology. This needs to be taken into consideration when interpreting the images and for fracture assessment the fat- and water-separated images as well as SW-like images should be evaluated simultaneously to avoid false-positive results.B0 inhomogeneities when compared to Cartesian or radial imaging. The use of artifact reduced UTE sequences on 3-T and 1.5-T MR systems needs to be assessed in the future.Another limitation was the use of phase information in the weighting of the SWI magnitude, which causes not only the osseous structures to be weighted but also parts with high fat content. Therefore, SW-like images comprised both susceptibility and chemical shift effects. In this study, an increased contrast of the vertebrae to the surrounding soft tissue was seen with increasing phase masks applied. Inversely a slight but non-significant decrease in the visibility of the thin cortical layer and fracture lines was detected most likely due to the increasing chemical shift effects. This needs to be considered when evaluating the SW-like images. Effects on the contrast of trabecular bone were not assessed as the primary focus was on the fracture detection and differentiation. Patients with metallic implants were not included in the study due to artifacts in 3-T scanners. Nevertheless, due to the half-radial readout of UTE sequences, UTE imaging is known to be less affected by artifacts due to implants and strong In summary, detection and age assessment of vertebral fractures as well as the morphological assessment of fractures and degenerative bone changes of the spine were feasible and accurate using water-fat-separated images as well as SW-like images with CT-like contrast, both derived from the sUTE-Dixon technique. This was possible by developing a new methodology which solved the smoothness-constrained non-linear inverse water-fat problem while at the same time removing the unwanted low-frequency phase terms. Simultaneously extracting water-fat images and CT-like images from one single MR sequence could be highly useful for clinical examinations due to a reduction of overall examination times and radiation exposure.


IntroductionNon-immersive virtual reality (NIVR) is emerging as an advantageous intervention in the arena of neurorehabilitation. Promising results have been obtained by the application of NIVR in adults with various chronic neurological conditions such as stroke and Parkinson’s disease, but studies on the use of NIVR in children with unilateral cerebral palsy (CP) are limited.Materials and methodsThis preliminary study included 10 school-aged participants with unilateral CP who were allocated into experimental and control groups. In accordance with the allocation ratio of 1:1, there were five participants in each group. During six weeks of intervention, children in the experimental group received NIVR intervention in addition to conventional physiotherapy, while those in the control group received only conventional physiotherapy, with a goal to improve hand function and functional independence. Nine-hole peg test (9HPT), box and block test (BBT), ABILHAND kids, and self-care section of functional independence measure for children (WeeFIM) were used as outcome measures.ResultsThere was significant improvement in all outcome measures in both groups. However, the improvement in the hand function and functional independence was significantly more in the experimental group than in the control group.ConclusionIt can be concluded that NIVR intervention in the management of children with unilateral CP seems to be feasible and useful. Further research with a larger sample size must be undertaken to reinforce these preliminary findings. Non-immersive virtual reality (NIVR) is emerging as a means of intervention in the arena of neurorehabilitation. NIVR has been found to be beneficial in the rehabilitation of the geriatric population ,2. PromiChildren with unilateral CP usually tend to avoid using the hand on the affected side, leading to dependence for activities that need bilateral hand usage. Consequently, there is increased burden of care on caregivers. This study aims to investigate the effect NIVR on hand function and functional independence in children with unilateral CP.Ref.No. DMIMS(DU)/IEC/2020-21/131 and was executed in conformation to the Declaration of Helsinki. This pilot study is an interventional, non-randomized trial with an active control group. The inclusion criteria comprised age between 6 and 12 years, diagnosis of unilateral CP, levels I-III on Gross Motor Function Classification System (GMFCS), and levels I-III on Manual Ability Classification System (MACS). Exclusion criteria included epilepsy, surgery in the past six months, Botox treatment in the past three months, and inability to understand commands.The study was conducted at Neurosciences Centre, Acharya Vinoba Bhave Rural Hospital, Datta Meghe Institute of Medical Sciences (DMIMS), Wardha, Maharashtra, India. The study was approved by the Institutional Ethical Committee of DMIMS with the approval number Screening for eligibility criteria was done. A parent or legal guardian of each participant signed the informed consent form. After the baseline assessment, this preliminary study recruited 10 participants with unilateral CP who were allocated into the experimental group (group A) and control group (group B). In accordance with an allocation ratio of 1:1, there were five participants in each group.The duration of each session was 60 minutes. The children in the experimental group underwent 30 minutes of NIVR-based intervention using a driving simulation game with PlayStation 4 , as shown in Figure There was no significant difference in the age-wise distribution of participants between group A  and group B (control), as shown in Table There was no significant difference in the gender-wise distribution of participants between group A  and group B (control), as shown in Table By using the chi-square test, statistically, no significant difference was found in the GMFCS level among participants in group A and group B , as shown in Table By using the chi-square test, no statistically significant difference was found in the MACS level among participants in group A and group B , as shown in Table Mean 9HPT score in the children of group A was 55.80±6.01 pre-treatment and it was 39.80±4.43 post-treatment. By using Student’s paired t-test, a statistically significant difference was found between pre-test and post-test 9HPT scores , as shown in Table Mean pre-treatment 9HPT score of the children in group A was 55.80±6.01 and in group B it was 56.80±7.19. By using Student’s unpaired t-test, no statistically significant difference was found in pre-treatment 9HPT scores between group A and group B , as shown in Table Mean BBT score in the children of group A was 15.60±3.50 pre-treatment and it was 26.60±2.30 post-treatment. By using Student’s paired t-test, a statistically significant difference was found between pre-test and post-test BBT scores , as shown in Table Mean pre-treatment BBT score of the children in group A was 15.60±3.50 and in group B it was 14±3.53. By using Student’s unpaired t-test, no statistically significant difference was found in pre-treatment BBT scores between group A and group B , as shown in Table Mean ABILHAND kids score in the children of group A was 50.40±6.54 pre-treatment and it was 64.00±3.00 post-treatment. By using Student’s paired t-test, a statistically significant difference was found between pre-test and post-test ABILHAND kids scores , as shown in Table Mean pre-treatment ABILHAND kids score of the children in group A was 50.40±6.54 and in group B it was 44.40±7.36. By using Student’s unpaired t-test, no statistically significant difference was found in pre-treatment ABILHAND kids scores between group A and group B , as shown in Table Mean WeeFIM (self-care) score in the children of group A was 28.60±7.36 pre-treatment and it was 35.40±7.23 post-treatment. By using the Wilcoxon signed rank a test, a statistically significant difference was found between pre-test and post-test WeeFIM (self-care) scores , as shown in Table Mean pre-treatment WeeFIM (self-care) score of the children in group A was 28.60±7.36 and in group B it was 25.80±5.80. By using the Mann-Whitney U test, no statistically significant difference was found in pre-treatment WeeFIM (self-care) scores between group A and group B , as shown in Table Statistical analysis was conducted by descriptive and inferential statistics using the chi-square test, Student’s paired and unpaired test, Wilcoxon signed rank test, and Mann-Whitney U test, software used in the analysis were SPSS 27.0 version  and GraphPad Prism 7.0 version, and p<0.05 was considered as a level of significance.The protocol of this study has been adapted from a previously published study , though The principles of neuroplasticity and that of motor learning including explicit feedback and multimodal stimulation are well tapped by virtual reality (VR) systems . BesidesImmersive VR systems use head-mounted display  that mayApart from neurorehabilitation, VR has been utilized as a distraction for pain management in children ,27. VR-bIt can be concluded that the study design is feasible and can be used with a larger sample size for further trial. The preliminary findings of this study, although limited by a small size of the sample, indicate that NIVR deserves exploration as a viable intervention for improving hand function and for decreasing dependence in the performance of routine activities for children with unilateral CP. NIVR came out as an interesting way to engage children with unilateral CP in an activity that requires bilateral hand use, which they otherwise avoided.


The gut microbiota profile is unique for each individual and are composed by different bacteria species according to individual birth-to-infant transitions. In the last years, the local and systemic effects of microbiota on cancer onset, progression and response to treatments, such as immunotherapies, has been extensively described. Here we offer a new perspective, proposing a role for the microbiota based on the molecular mimicry of tumor associated antigens by microbiome-associated antigens.In the present study we looked for homology between published TAAs and non-self microbiota-derived epitopes. Blast search for sequence homology was combined with extensive bioinformatics analyses.Several evidences for homology between TAAs and microbiota-derived antigens have been found. Strikingly, three cases of 100% homology between the paired sequences has been identified. The predicted average affinity to HLA molecules of microbiota-derived antigens is very high (< 100 nM). The structural conformation of the microbiota-derived epitopes is, in general, highly similar to the corresponding TAA. In some cases, it is identical and contact areas with both HLA and TCR chains are indistinguishable. Moreover, the spatial conformation of TCR-facing residues can be identical in paired TAA and microbiota-derived epitopes, with exactly the same values of planar as well as dihedral angles.+ T cell responses are very likely induced. Therefore, the anti-microbiota T cell memory may turn out to be an anti-cancer T cell memory, able to control the growth of a cancer developed during the lifetime if the expressed TAA is similar to the microbiota epitope. This may ultimately represent a relevant selective advantage for cancer patients and may lead to a novel preventive anti-cancer vaccine strategy.The data reported in the present study show for the first time the high homology in the linear sequence as well as in structure and conformation between TAAs and peptides derived from microbiota species of the Firmicutes and the Bacteroidetes phyla, which together account for 90% of gut microbiota. Cross-reacting CD8The online version contains supplementary material available at 10.1186/s12967-022-03512-6. SeveralGut bacteria regulate food digestion along the gastrointestinal tract and prevent bacteria invasion by maintaining the intestinal epithelium integrity . In addiBesides the physiological role, the microbiota have a role in human diseases , 11 and In addition to such “generic” mechanisms of modulation, the impact of the gut microbiota on the anti-cancer immune response can be driven by a “molecular mimicry” between bacteria and tumor associated antigens.+ T cell receptor (TCR), given that a single TCR is cross-reactive recognizing at least 106 different MHC-bound peptides [http://www.cbs.dtu.dk/services/NetMHC/logos.php) [The gut microbiome encodes over 3 million genes as whole, whereas the entire human genome consists of approximately 23,000 genes . Therefopeptides , 27. Indgos.php) , 29. Thehttps://caped.icp.ucl.ac.be/Peptide/list) share sequence homology to viral sequences [+ T cells cross-reacting with tumor antigens, able to control the growth of a cancer lesion, if expressing a TAA similar to the viral epitope. This may ultimately represent a relevant selective advantage for cancer patients and may lead to a novel preventive anti-cancer vaccine strategy [Based on this assumption, we have recently shown that TAAs .All the peptides selected in the study were predicted with the NetMHCpan 4.1 and the NetMHCstabpan 1.0 predictive algorithms (https://caped.icp.ucl.ac.be/Peptide/list) were used to interrogate NetMHCpan 4.1 tool. Nanomer peptides for the MHC class I HLA-A* 0201 and 2402 alleles  have been selected with a predicted affinity value < 100 nM .The peptides deposited at the cancer antigenic peptide database . Homologous microbiota protein sequences have been extracted from the protein database of the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/) and epitope prediction has been performed with the NetMHCstabpan 1.0 tool.The TAAs selected as SB according to NetMHCpan 4.1 prediction tool have been submitted to BLAST for a protein homology search against Firmicutes (taxid:1239) and Bacteroidetes (taxid:976) sequences within the non-redundant protein sequences database (https://www.rcsb.org) for the HLA-A*02:01 and HLA-A*24:02 molecules. The 1AO7 complex was selected which includes the structure of the HTLV-I LLFGYPVYV epitope crystallized with the HLA-A*02:01 molecule, the β2 microglobulin, the α and β chains of the TCR (PDB https://www.rcsb.org/structure/1AO7). The 7JYV complex was selected which includes the structure of the influenza YFSPIRVTF epitope crystallized with the HLA-A*24:02 molecule (https://www.rcsb.org/structure/7JYV). The PyMol software  was used to modify the epitope sequences into the peptides analyzed in the present study. The Molsoft Mol Browser (version 3.8-7d) was used to generate the epitope modelling and molecular docking as well as to calculate the inter-atom planar and dihedral angles.Specific reference structures were selected from the protein data bank PBD  to the most frequent MHC class I alleles were selected, as previously described .Firmicutes and Bacteroidetes phyla within the GeneBank non-redundant protein database. The search returned a large number of HLA-A*0201 and 2402 restricted bacterial sequences sharing homology with selected TAAs and the majority of such homology was found for the HLA-A*0201. Indeed, 267 and 159 homologous peptides were identified for Firmicutes and Bacteroidetes, respectively, for HLA-A*02:01. Of these, 95 and 55 were identified with a high affinity value < 10 nM for Firmicutes and Bacteroidetes, respectively. The MAGE-A10 was the TAA with the highest numbers of homologous peptides for both Firmicutes and Bacteroidetes (e.g. 50 and 28) while the MAGE-C1 and MAGE-C2 were the TAA with the lowest numbers of homologous peptides for Firmicutes and Bacteroidetes (e.g. 12 and 10), respectively. On the contrary, 112 and 81 homologous peptides were identified for Firmicutes and Bacteroidetes, respectively, for HLA-A*24:02. Of these, none and only 1 were identified with a high affinity value < 10 nM for Firmicutes and Bacteroidetes, respectively. The SAGE and the KM-NH-1b were the TAA with the highest numbers of homologous peptides for Firmicutes and Bacteroidetes (e.g. 38 and 24), respectively; while the MAGE-A2 and SAGE were the TAA with the lowest numbers of homologous peptides for Firmicutes and Bacteroidetes (e.g. 18 and 18), respectively  showed 6 or 7 residues along the sequence identical to the corresponding TAA Table .Table 1AStrikingly, three microbiota-derived peptides showed a sequence identical to the corresponding TAA, the GnTV (Firmicutes Oscillospiraceae bacterium Seq ID MBP0955392.1), MAGE-C1 (Firmicutes Clostridia bacterium Seq ID MBO5479965.1) and MK-NH-1 (Firmicutes Roseburia sp. 1XD42-69 Seq ID WP_120409454.1)  Table .Indeed, the average percentage of identical residues at each position is 82.8%, ranging from 79.7% at position 3 to 89.5% at position 2. Summing up also the conservative substitutions, the average percentage of at each position is 93.6%, ranging from 86.7% at position 7 to 98.3% at position 9. Consequently, the average percentage of non-conservative substitutions at each position is only 6.39%, ranging from 1.6% at position 9 to 13.2% at position 7. In such analysis, position 1 represents an outlier with the far lowest average percentage of identical 48.2%) and identical + conservative substitutions (62.4%) and the far highest average percentage of non-conservative substitutions (37.6%)  than for HLA*24:02   strikingly similar to the corresponding TAA. Indeed, in some cases, the structures of paired TAA and microbiota-derived epitopes are indistinguishable Fig. A and B.FAdditional information about the similarity between the paired TAA and microbiota-derived epitopes is provided by the peptide backbones. Indeed, considering the spatial position of the residues interacting with the TCR, it is possible to identify paired epitopes which share the same planar as well as dihedral angles ± 10%) between selected atoms of the amino acid residues in the same position of the peptide. Such analysis shows that, among paired TAA and microbiota-derived epitopes with highly similar structures, it is possible to identify selected pairs with 100% identical angle values, especially in HLA-A*02:01 epitopes . To further confirm the biological relevance of such a molecular mimicry, the consensus of the microbiota-derived epitopes always perfectly matches with the sequence of the corresponding TAA frequent residues. Moreover, the amino acid substitutions at each position are mostly conservative, confirming that the replacement does not significantly impact on the charge and conformation of the peptide structure.The number of homologous peptides with high affinity (< 10 nM) to HLA-A*02:01 molecule indicates that the molecular mimicry between TAA and microbiota-derived epitopes is not anecdotal and suggest a potential relevance in cross-reacting T cell response. In particular, about 70% of the paired epitopes show 6–7 identical residues along the 9-aa peptide sequence, 7% of them shows 8 identical residues and three of them show an identical sequence. Such long stretches of identical amino acids in a nonamer sequence suggest a great biological relevance given that a random event of an identical stretch of 6–9 amino acid in a nonamer sequence has an extremely low probability to occur  and in about 50% of cases this is even higher than the corresponding TAA. According to our previous studies, such affinity values have a 100% correspondence to experimentally validated binding to HLA molecules. Therefore, this supports the concept that such microbiota-derived epitopes are indeed efficiently presented on the surface of cells to elicit a T cell response. Finally, the structural conformation of the microbiota-derived epitopes is, in general, highly similar to the corresponding TAA. In some cases, it is identical and contact areas with both HLA and TCR chains are indistinguishable. The spatial conformation of TCR-facing residues can be identical in paired TAA and microbiota-derived epitopes, with exactly the same values of planar as well as dihedral angles. This confirms that specific amino acid substitutions in the linear sequence do not affect the peptides structure and conformation which can be recognized by cross-reacting T cells. In support to this hypothesis, we have recently shown that CD8nt study .The biological relevance of the findings herein described is that, depending on the stage of the human life when the individuals will encounter the microbiota species, such homology may represent a favorable or unfavorable factor. Indeed, if the microbiota species have colonized the gastrointestinal tract in the first few months of life, the immune system could recognize the derived peptides as self-antigens and the specific T cell clones would be removed. In this case, a tumor lesion presenting a homologous TAA would have a selective advantage for establishing and progressing with a poor prognosis.On the contrary, if the microbiota species have colonized the gastrointestinal tract later during life, the immune system could recognize the derived peptides as non-self-antigens and specific memory T cell clones would be established. In this case, as for a preventive vaccine, a tumor lesion presenting a homologous TAA would promptly recall the memory T cells able to eliminate or control tumor growth with an improved prognosis.The relevance of our findings resides in the fact that the microbiota-derived antigens show homology with TAAs shared by several cancers of different histological origin. In particular, MAGE-A3 and MAGE-A10 have been identified in non-small cell lung cancers (NSCLC), bladder cancers, esophageal and head and neck cancers, and sarcomas . In partThese findings open a new horizon in the mutual interaction between the microbiota and immune response in humans with a potential profound impact on tumor development and progression. Moreover, they provide a completely novel class of antigens to be possibly used as anti-cancer preventive vaccination, even administered as food integrators.If confirmed, this would completely revolutionize the cancer immunotherapy field and represent a milestone in fighting cancer.Additional file 1: Figures S1–4. Percentage (average) of type of residues at each position of the microbiota-derived epitopes when aligned to the corresponding TAA. (A) Identical residues; (B) conservative residues; (C) non-conservative residues; (D) Identical + conservative residues. Figure S5. Affinity to HLA-A* 02:01 and 24:02 molecules of each microbiota-derived epitopes with homology to the corresponding TAA. The red dotted line indicates the affinity of the TAA. Figure S6–17. Predicted 3D conformation of TAA and microbiota-derived paired peptides. The surface conformation of the paired TAA microbiota-derived peptides is shown. Residues in the microbiota-derived epitopes identical to the TAA sequences are indicated in green color. Green areas = contact points with HLA-A molecule; Magenta areas = contact points with TCR α chain; Light Blue = contact points with TCR β chain. The latter are available only in Figure S6–S13. (HLA-A* 02:01 restricted epitopes). Figures S18. Percentage of identical values (± 10%) of planare and dehidral angles between of the microbiota-derived epitopes and the corresponding TAA. Figures S19–S24. Structural predicted backbone conformation of paired TAA and microbiota-derived epitopes. Green areas = contact points with HLA molecule; Magenta areas = contact points with the TCR α chain; light blue areas = contact points with the TCR β chain. The latter are available only in Additional file Figure S19–22. (HLA-A* 02:01 restricted epitopes). Values of the planar as well as dihedral angles between atoms of selected TCR-facing residues are shown in parallel.


There was a significant decrease in AA in both groups between preoperatively and at 1-week  and 1-month follow-up . For the rest of the binocular measurements, no statistically significant postoperative changes were found in any group. This finding suggests follow-up studies on amplitude of accommodation in phakic intraocular lens ICL implantation.The aim was to evaluate accommodative and binocular function of phakic intraocular lens implantable collamer lens (ICL) in high and low-to-moderate myopia. Prospective comparative cohort study with 38 myopic patients who underwent ICL implantation were divided into two groups of 19 patients, each one based on the spherical equivalent (SE): high-power (SE ≤ −6 D) and low-to-moderate (SE > −6 D). The push-up amplitude of accommodation (AA), monocular accommodative facility (MAF), distance and near ocular deviation, near convergence amplitude, near point convergence (NPC), stereopsis, and accommodative convergence/accommodation (AC/A) ratio were assessed before surgery and 1 week and 1 month postoperatively. The mean residual refractive error at 1 month after surgery improved in both groups, 0.18 ± 0.34 D and 0.09 ± 0.26 D, respectively ( There is a variety of refractive procedures to correct myopia, such as corneal refractive surgery, crystalline-lens replacement with an intraocular lens (IOL), or phakic intraocular lens (P-IOL) implantation. One of the most widely-used and worldwide-used p-IOL types is the implantable collamer lens , and the outcomes reported in a recent review for myopia correction support its use, confirming the safety, efficacy, and predictability of the procedure ,2,3.In any case, although there are many publications showing the outcomes obtained with p-IOL ICL, the references are very limited regarding the evaluation of accommodative and binocular function after the implantation in combination ,4,5.It is important to emphasize that myopic patients tend to accommodate less than emmetropes for all target distances when wearing distance corrective lenses , and p-IConsequently, the accommodative and binocular vision evaluation could be relevant because the refractive change is very high in most of the patients, and there can be a high anisometropia due to the delay between the implantation of the first and the second lens that could decompensated their binocular vision. There is no evaluation comparing the accommodative and binocular changes between patients with different degrees of myopia implanted with these lenses.Therefore, the aim of this study is to analyze the changes and effect of bilateral implantation of p-IOL ICL on accommodative and binocular function for high and low-to-moderate myopia.® p-IOL ICL™  implantation by an expert surgeon (FP) from February 2019 to December 2020. They were divided into two groups based on baseline spherical equivalent (SE) obtained after subjective refraction: the high-power group (SE ≤ −6 D) and low-to-moderate power group (SE > −6 D) [This prospective and controlled study was conducted at Miranza IOA Clinic Madrid. It included 38 myopic patients who underwent VisianThe sample size was exclusively calculated based on amplitude of accommodation at one month after surgery, which is the main variable of the study, to detect a difference equal or greater than 2 D, assuming a standard deviation of 2.72 according to the results related to the push-up AA at one month after surgery published by Kamiya et al.  acceptinInclusion criteria were myopia from −0.75 to −20 D and regular astigmatism lower than 1.50 D, age from 18 to 40 years, corrected distance visual acuity (CDVA) better than 20/25, and patients who did not wear contact lenses during the six previous months prior to recruitment. Exclusion criteria were presence of manifest tropia, a history of strabismus surgery, anisometropia greater than 1.5 D, accommodative dysfunction, previous strabismus or intraocular surgery, absence of binocular vision, anterior segment pathologic conditions, and systemic pathologies such as neurological or vascular diseases or pharmacological treatment  with side effects on accommodative function.All patients provided written informed consent. The study was approved by the local ethics committee, and it was performed in accordance with the Declaration of Helsinki. The study was approved by the ethic committee Clínico San Carlos Hospital, Madrid, Spain (CI. 18/493-E).® p-IOL ICL™ , the pupil was dilated 30 min before surgery. After injecting 1% sodium hyaluronate into the anterior chamber, the ICL was implanted in the posterior chamber through a 2.8 mm temporal corneal incision. The following step was to wash away the viscoelastic surgical agent using a balanced salt solution, and a miotic agent (acetylcholine) was then instilled. Postoperative medications included topical antibiotics and steroids. All the surgeries were performed without complications.For surgery with the Visian®  and swept-source IOL Master® 700 biometer. The size of the ICL was calculated with the calculator developed by the manufacturer (https://ocos.staarag.ch/landing/ accessed on 3 February 2020). The target refraction was based on emmetropia.For the calculation of the lens power required for each eye, the data taken into account were the patient’s subjective refraction, the anterior chamber depth, keratometry, the white-to-white distance, and the corneal pachymetry measurements, which were performed by corneal tomography Pentacam HR®.All patients underwent a complete ophthalmological examination before surgery, including a detailed anamnesis, uncorrected distance visual acuity (UDVA) and CDVA measured with a Snellen chart, manifest and cycloplegic refraction (three drops of tropicamide 1% instilled every 5 min and performing the refraction 30 min after the last instillation), slit lamp biomicroscopy, and fundus examination. In addition, corneal topography and biometric measurements were performed with the Pentacam HRAccommodative function was assessed by measuring the monocular amplitude of accommodation (AA) with the push-up method  and the Binocular vision evaluation was based on the results of extraocular motility testing, ocular deviation for distance and near fixation, fusional vergence for near fixation, near point of convergence (NPC), stereopsis, and accommodative convergence/accommodation (AC/A) ratio. In addition, the convergence insufficiency symptom survey (CISS-V15) was assessed according to the authors methodology to quantify associated symptomatology ,13. Ocular deviation was measured with a prism and alternate cover test (PACT) at distance and near fixation . FusionaPostoperative visits were performed at 1 week and 1 month after surgery. Follow-up examinations included a full ophthalmological examination carried out by the surgeon and accommodative and binocular assessment performed by trained optometrists.Accommodative and binocular tests were performed with the best manifest refraction placed in trial frames. t-test) or parametric (Wilcoxon signed-rank) tests. The p-value threshold for statistical significance was set at 0.05.The main focus of the study was to evaluate binocular vision; therefore, all binocular variables of all patients were included for the analysis. For the monocular variables, only data from the right eyes were presented ,18. For p > 0.05). Demographic data are summarized in p < 0.001) in the high-power group and 0.03 ± 0.12 D (p < 0.001) in the low-to-moderate group one week postoperatively; 0.18 ± 0.34 D (p < 0.001) and 0.09 ± 0.26 D (p < 0.001), respectively, at one month. All postoperative refractive error values were statistically significant different from preoperative values (p < 0.05). The study enrolled 38 patients, and the mean age was 29.8 ± 4.7 years (range 21 to 38 years). The high-power group included 19 patients (53.3% men and 46.7% women), and the mean age was 30.0 ± 5.1 years, and the low-to-moderate-power group included 19 patients (47.4 % men and 52.6% women), and the mean age was 29.6 ± 4.4 years (range 22 to 36 years) There were no differences in age between groups  and one-month postoperative visit  (The preoperative AA was 11.38 ± 2.82 D (range 7.40 to 18.18) and 10.70 ± 2.16 D  in the high-power group and in the low-to-moderate group, respectively There was a significant decrease in AA in both groups between preoperatively and one-week follow-up (ctively) . Insteadctively) .The extraocular motility examination showed no cases of vertical deviation or decompensated phoria in any of the groups. The mean preoperative stereopsis values (seconds of arc) and one-month postoperative was 41.00 ± 24.93 and 31.19 ± 12.29, respectively, in the high-power group and 41.26 ± 24.25 and 33.97 ± 13.93, respectively, in the low-to-moderate group. There were no statistically significant differences between the visits in both groups.p < 0.001; p = 0.001, respectively) and one-month postoperative visits . The NPC break and NPC recovery in the high-power group showed a statistically significant increase one week after the surgery ; however, both values reverted to pre-op values one month after surgery. The NPC recovery in the low-to-moderate group showed a significant increase one week after surgery (p = 0.036) although it went back to normal one month after surgery  induces an increase in the accommodative demand , and perAnother test that evaluates the accommodative function is the MAF, which assesses the ability to change focus quickly (far–near). We could expect that this measurement would also be altered after ICL implantation; however, changes have not been shown. Specifically, a slight increase in its value has been observed, meaning a better ability to make far–near focus changes although without statistical significance. On the other hand, other authors ,22 with Moreover, binocular vision evaluation can be relevant to reinforce the knowledge about binocular changes after p-IOL implantation. The abrupt emmetropization after p-IOL implantation not only changes the accommodative demand but also leads to a sudden, higher convergence demand in near-vision tasks . To respond to the main objectives of the study, different binocular variables were analyzed, and we found no changes in any of them except in the NPC blur point.Distance and near ocular deviation in the high myopia group tends to ortophoria after surgery. Similar results were reported by Kato et al., finding changes in distance and near ocular deviation that were less exophoric than pre-op values .Near convergence amplitude, break and recovery points showed no changes after surgery, and there were no differences between groups at either one-week or one-month visits . The tenAC/A ratio was similar after p-ICL implantation during the first month after surgery in both groups. These results are in line with the outcomes reported by Chen et al. and Luo et al. ,22. On tPhakic IOL implantation has demonstrated high optical quality and potential visual acuity improvement in myopic patients due to retinal magnification , so thisThe NPC is a basic metric to report accommodative and binocular function. In the current study, no differences were found in any NPC values except a deterioration in NPC blur  in both low-to-moderate and high myopia group at one week and one month after surgery . These rDespite changes in AA and NPC blur one month after p-IOL implantation in both myopic groups, there were no differences in symptoms perceived by patients, according to the CISS-V15. Due to the relationship between accommodation and convergence, a decrease in accommodation capacity (identified by a decrease in AA) could trigger a decrease in convergence capacity and an increase in near exophoria, leading to a situation that could decompensate the binocular vision of a healthy patient. Our results show a decrease in the accommodation; however, the convergence has not been altered. After p-IOL implantation, patients did not become more exophoric at near distance, and they did not suffer a worsening of the convergence reserves, the level of stereopsis did not decrease but even slightly improved, and the values of the near point of convergence did not worsen.The current study has some limitations. One of them is the short follow-up time, and the other is the use of a subjective method for the measurement of the accommodative function. It is necessary to move towards objective methods of measuring accommodation.In conclusion, p-IOL ICL implantation did not modify baseline accommodative and binocular vision after the surgery in healthy patients with low-to-moderate or high myopia groups except a decrease in the amplitude of accommodation although this change was not clinically relevant due to the age of the patients included in this study. Future studies would be needed to know the impact of this decrease in pre-presbyopes and emerging presbyopes.